斜卧青霉L-06内切葡聚糖酶Ⅰ基因的克隆与表达  

Cloning and expression of egI gene from Penicillium decumbens L-06

作　　者：刘韫滔[1] 韩学凤[1] 罗泽宇[1] 邱玉峰[1] 龙敏南[2] 胡忠[1] 

机构地区：[1]汕头大学生物学系,广东汕头515063 [2]厦门大学能源研究院,福建厦门361005

出　　处：《微生物学通报》2012年第5期696-701,共6页Microbiology China

基　　金：国家自然科学基金项目(No.41076106);广东省自然科学基金项目(No.S2011030005257);广东省科技计划项目(No.2009B090300346);广东高校科技创新重点项目(No.CXZD1124)

摘　　要：【目的】克隆斜卧青霉L-06的内切葡聚糖酶Ⅰ基因(egI),并实现其在大肠杆菌内的高效表达。【方法】利用RT-PCR技术克隆了斜卧青霉L-06的内切葡聚糖酶Ⅰ基因(egI),并将egI基因克隆到原核表达载体中,构建了重组质粒pET32a-egI。【结果】转化至大肠埃希菌Rosetta(DE3),经IPTG诱导重组蛋白表达,SDS-PAGE检测结果表明:重组表达产物的相对分子质量约为80 kD,与预期相符。重组表达的菌悬液,经破碎离心,取其上清液,进行纤维素酶活性染色,获得了活性条带。DNS法测得内切酶活力为2.56 IU/mL。【结论】构建了斜卧青霉L-06内切葡聚糖酶Ⅰ的原核表达系统。[Objective] The research focus on cloning endoglucanase I（egI） gene from Peni-cillium decumbens L-06 and expressing in Escherichia coli with high efficiency.[Methods] egI gene was cloned from Penicillium decumbens L-06 by RT-PCR method.Recombinant plasmid pET32a-egI was constructed and was transformed into Escherichia coli rosetta（DE3）.Recombinant protein with His-tag was expressed in E.coli rosetta（DE3） after induction with IPTG and then was purified with the Ni-NTA affinity chromatography.[Results] As expected,the relative molecular mass was approximately 80kD after analyzed by SDS-PAGE and Western blotting.Hydrolysis activity of recombinant protein was assayed by cellulase activity staining and DNS method（2.56 IU/mL）.[Conclusion] The results achieve the purpose as con-structing prokaryotic expression system and expressing egI gene.

关 键 词：斜卧青霉L-06 内切葡聚糖酶Ⅰ 克隆 原核表达 

分 类 号：Q78[生物学—分子生物学]